Nicotine at physiologic concentrations impairs macrophage killing of Mycobacterium tuberculosis (MTB), resulting in a 2-5 fold increase in bacterial burden. We have also found that preventing nicotine binding ? either pharmacologically or by genetic disruption of the nicotinic receptor ? significantly mitigated cigarette smoke extract-induced impairment of macrophage killing of MTB. But the mechanisms by which nicotine impairs macrophage control of MTB are not known. Our hypothesis is that nicotine sabotages macrophage function against MTB (i) directly through NF?B-mediated inhibition of autophagy and apoptosis as well as through DICER/microRNA-mediated inhibition of host-protective cytokine production and (ii) indirectly through increased production of CTLA-4 and activation of T regulatory cells (Tregs). Aim 1: Determine the mechanisms by which nicotine directly impairs macrophage killing of MTB. Approach: We will isolate nicotine-nave human alveolar macrophages (AM) and monocyte-derived macrophages (MDM), infect them with MTB nicotine, and quantify autophagy, apoptosis, intracellular burden of MTB, and the extent NF?B inhibition mitigates the effects of nicotine. We will also knockdown specific NF?B subunits and DICER to determine their roles in nicotine-mediated inhibition of anti-MTB immunity. Hypothesis: Nicotine induction of NF?B in AM and MDM will inhibit autophagy and apoptosis, resulting in increased burden of MTB. Inhibition of NF?B activation will abrogate these effects of nicotine and restore macrophage killing of MTB. Knockdown of DICER will reverse the suppression of MTB-induced cytokines by nicotine. Aim 2: Determine the mechanisms by which nicotine indirectly impairs macrophage killing of MTB. Subaim A. An ex vivo model using primary human cells. Approach: We will culture nave human MDM with nave vs. nicotine-exposed Tregs and infect the cells with MTB. To determine if CTLA-4 expression is responsible for nicotine-induced increase in Treg activity and secondary MDM suppression, the cells will also be incubated with anti-CTLA-4 neutralizing antibody. Hypothesis: Nicotine will increase CTLA-4 expression on Tregs, augmenting their production of IL-10 and TGF?. This will inhibit autophagy in MTB-infected MDM and result in greater bacterial burden compared to MDM co-cultured with nicotine-nave Tregs. Antagonism of CTLA-4 will abrogate these immunosuppressive effects of nicotine. Subaim B. In vivo murine model. Approach: We will adoptively transfer Tregs from unexposed or nicotine- exposed B6.PL(Thy1.1) mice into Treg-depleted Foxp3+GFP+DTR+(Thy1.2) mice, infect the recipient mice with MTB, and quantify MTB burden, macrophage and T cell phenotypes, lung histopathology, and survival. Hypothesis: Mice receiving nicotine-exposed Tregs will have fewer host-protective M1 lung macrophages and TH1 and TH17 cells compared to mice receiving nicotine-nave Tregs. As a result, mice receiving nicotine- exposed Tregs will be more susceptible to MTB, demonstrated by greater MTB burden, fewer host-protective macrophages and T helper phenotypes, more severe lung pathology, and decreased survival. Potential impact of project: U.S. Veterans have a high prevalence of nicotine exposure (through use of cigarette smoke and smokeless nicotine products) and a greater risk for TB due to overseas deployment in TB endemic countries, relatively high prevalence of homelessness, and advancing age. While nicotine impairs human macrophage control of MTB, no work has been conducted to determine how this occurs; illuminating these mechanisms will provide the scientific impetus to help alert the medical and public health communities of this danger of concomitant nicotine and MTB exposures in veterans and non-veterans. Findings from these studies can also provide the foundation for developing immunomodulatory approaches to treatment even in the absence of nicotine exposure; e.g., use of clinically available anti-CTLA-4 agents to deactivate Tregs in an attempt to optimize host immunity against TB.